Dengue virus (DEN), a Category A priority pathogen, causes the most prevalent arthropod-borne viral illnesses in humans worldwide. Like other viruses, DEN lacks its own translational machinery and thus is dependent on the host cell for translation of viral RNA. DEN and other flaviviruses have a capped, nonpolyadenylated, positive-sense RNA genome. In contrast to many animal viruses, flaviviruses compete efficiently for the cellular translation apparatus without shutting off host protein synthesis. Our long-term goal is to define the mechanism of flavivirus translation, using DEN as a model, and to characterize the viral and cellular factors involved. Specifically, the proposed research investigates the sequence and structural determinants in the DEN 3' untranslated region (UTR) that stimulate viral translation and the factors that mediate this process. We have shown that the 3'UTR of the DEN genome stimulates translation in a manner similar to that reported for cellular and other viral systems but not yet for flaviviruses. In addition, we have identified sequence elements in the 3'UTR that modulate translation efficiency. The DEN 3'UTR can stimulate translation in the absence of viral proteins, leading to the hypothesis that the DEN 3'UTR stimulates viral translation by recruiting cellular factors. This will be addressed via the following two specific aims: Specific Aim 1: Characterize the role of the 3'UTR in stimulation of DEN translation and identify the sequences and structures in the DEN 3'UTR critical for viral translation. We have shown that the DEN 3'UTR acts as a translational enhancer in reporter constructs in both transfected cells and in vitro translation extracts and have begun to analyze sequences that contribute to this effect. Specific Aim 1 will focus on further delineation of sequences and structures in the DEN type 2 (DEN2) 3'UTR that regulate translation in the context of reporter constructs, DEN2 replicons and a DEN2 infectious clone. Specific Aim 2: Identify and characterize proteins that mediate 3'UTR-stimulated DEN translation. This aim focuses on isolation, identification, and characterization of the proteins that mediate viral translation via interaction with the DEN 3'UTR to begin to understand their mechanism of action. This work will provide insight into translation of DEN and other flaviviruses as well as into eukaryotic translational control. Moreover, these studies should identify targets for rational drug and vaccine development of this medically important pathogen.